• Journal of Ginseng Research
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• v.44 no.3
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• pp.475-482
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• 2020

Background: Active natural ingredients, especially small molecules, have recently received wide attention as modifiers used to treat neurodegenerative disease by promoting neurogenic regeneration of neural stem cell (NSC) in situ. 20(S)-protopanaxadiol (PPD), one of the bioactive ingredients in ginseng, possesses neuroprotective properties. However, the effect of PPD on NSC proliferation and differentiation and its mechanism of action are incompletely understood. Methods: In this study, we investigated the impact of PPD on NSC proliferation and neuronal lineage differentiation through activation of the Wnt/glycogen synthase kinase (GSK)-3β/β-catenin pathway. NSC migration and proliferation were investigated by neurosphere assay, Cell Counting Kit-8 assay, and EdU assay. NSC differentiation was analyzed by Western blot and immunofluorescence staining. Involvement of the Wnt/GSK3β/β-catenin pathway was examined by molecular simulation and Western blot and verified using gene transfection. Results: PPD significantly promoted neural migration and induced a significant increase in NSC proliferation in a time- and dose-dependent manner. Furthermore, a remarkable increase in anti-microtubule-associated protein 2 expression and decrease in nestin protein expression were induced by PPD. During the differentiation process, PPD targeted and stimulated the phosphorylation of GSK-3β at Ser9 and the active forms of β-catenin, resulting in activation of the Wnt/GSK-3β/β-catenin pathway. Transfection of NSCs with a constitutively active GSK-3β mutant at S9A significantly hampered the proliferation and neural differentiation mediated by PPD. Conclusion: PPD promotes NSC proliferation and neural differentiation in vitro via activation of the Wnt/GSK-3β/β-catenin pathway by targeting GSK-3β, potentially having great significance for the treatment of neurodegenerative diseases.

• Korean Journal of Veterinary Research
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• v.29 no.3
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• pp.283-289
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• 1989

In order to measure in vitro cell mediated immunity in the guinea pigs sensitized with the killed bacilli of Mycobacterium bovis ($AN_5$), M avium (serotype 2), M tuberculosis and M intracellulare (serotype 8), leukocyte adherence inhibition (LAI) test was established using the antigens of purified protein derivatives (PPD) tuberculin. By using LAI test, specificity of cell-mediated immune responses of the guinea pigs inoculated with various Mycobacterium spp was investigated, and comparison between values of LAI and skin test was also made to evaluate the specificity of the newly designed test. The results obtained throughout the experiments were summarized as follows; 1. The optimal concentration of PPD antigens for LAI test was 1 to 2mg per ml of medium. 2. When the leukocytes of guinea pigs sensitized with both M bovis($AN_5$) and M avium (serotype 2) for 2 to 8 weeks were incubated with homologous or heterologous PPD antigens, mean values of LAI test were $61.2{\pm}11.2$ and $65.6{\pm}5.1%$ in homologous PPD antigens respectively, while $30.0{\pm}3.7$ and $32.8{\pm}5.7%$ in heteNlogous PPD antigens, showing the prominently high value of LAI in the homologous syst,em (p<0.01). 3. When the leukocytes of guinea pigs sensitized with both M tuberculosis and M intracellulare (serotype 8) for 2 to 8 weeks were incubated with homologous and heterologous PPD antigens, mean values of LAI test were $67.9{\pm}2.9$ and $66.9{\pm}5.0%$ in homologous PPD antigens, while $27.4{\pm}7.4$ and $24.4{\pm}7.1%$ in heterologous PPD antigens, showing the prominently high value of LAI in the homologous system (p<0.01). 4. Comparing with the specificity of LAI and skin tests on the basis of the value obtained from the homologous system, deviation of reaction was revealed to be 49.5 to 100.2 in LAI test, and -15.9 to 52.0 in skin test.

• IMMUNE NETWORK
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• v.3 no.3
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• pp.235-241
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• 2003

Background: The protective immunity against tuberculosis (TB) involves both CD4+ T cells and CD8+ T cells. In our previous study, we defined four Mycobacterium tuberculosis derived peptide epitopes specific for HLA-$A^*0201$ restricted CD8+ T cells ($ThyA_{30-38}$, $RpoB_{127-135}$, $85B_{15-23}$, $PstA1_{75-83}$). In this study, we investigated the immune responses induced by these peptide specific CD8+ T cells in latently and chronically infected people with TB. Methods: We characterized these peptide specific CD8+ T cell population present in PBMC of both TB patients and PPD+healthy people using IFN-${\gamma}$elispot assay, intracellular staining and HLA-A2 dimer staining. Results: The frequency of peptide specific CD8+ T cell was in the range of 1 to 25 in $1.7{\times}10^5$ PBMC based on ex vivo IFN-${\gamma}$ elispot assay, demonstrating that these peptide specific CD8+ T cell responses are induced in both TB patients and PPD+ people. Short term cell lines (STCL) specific for these peptides proliferated in vitro and secreted IFN-${\gamma}$ upon antigenic stimulation in PPD+ donors. Lastly, HLA-$A^*0201$ dimer assays indicated that $PstA1_{75-83}$ specific CD8+ T cell population in PPD+ healthy donors is heterogeneous since approximately 25~33% of $PstA1_{75-83}$ specific CD8+ T cell population in PPD+ healthy donors produced IFN-${\gamma}$ upon peptide stimulation. Conclusion: Our results suggest that MHC class I restricted CD8+ T cell mediated immune responses to M. tuberculosis infection are induced in both TB patients and PPD + people; however, the CD8+ T cell population is functionally heterogeneous.

• Journal of the Korean Chemical Society
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• v.18 no.5
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• pp.381-388
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• 1974

When mixture of p-phenylenediamine (PPD) and m-aminophenol (MAP) were reacted with formaldehyde (F) varying their amounts under $N_2$ stream at the temperature of -5∼0$^{\circ}$, addition condensation reaction occurred and brown colored resins(in some cases orange colored) were formed immediately. All resins thus formed were insoluble in most ordinary organic solvents and did not melt up to 300$^{\circ}$. When the resins were treated with dilute(7 %) aqueous sodium hydroxide solution, the adsorptivity of methylene blue on them showed marked improvement reaching as much as 80 mg of methylene blue on 1 g of the resin. On the other hand, in the case of bromophenol blue, its amount of adsorption appeared 250 mg per 1 g of the resin. The TGA under $N_2$ atmosphere indicated that the resin formed in molar ratio of 1 : 3 : 8 (PPD : MAP : F) showed the best heat-resistant property among others. About 40 % weight loss was observed for this resin at 900$^{\circ}$ with heating rate of 2$^{\circ}$ per minute.

• Journal of Ginseng Research
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• v.41 no.3
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• pp.392-402
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• 2017

Background: Previously, we reported that Korean Red Ginseng inhibited liver fibrosis in mice and reduced the expressions of fibrogenic genes in hepatic stellate cells (HSCs). The present study was undertaken to identify the major ginsenoside responsible for reducing the numbers of HSCs and the underlying mechanism involved. Methods: Using LX-2 cells (a human immortalized HSC line) and primary activated HSCs, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assays were conducted to examine the cytotoxic effects of ginsenosides. $H_2O_2$ productions, glutathione contents, lactate dehydrogenase activities, mitochondrial membrane permeabilities, apoptotic cell subpopulations, caspase-3/-7 activities, transferase dUTP nick end labeling (TUNEL) staining, and immunoblot analysis were performed to elucidate the molecular mechanism responsible for ginsenoside-mediated cytotoxicity. Involvement of the AMP-activated protein kinase (AMPK)-related signaling pathway was examined using a chemical inhibitor and small interfering RNA (siRNA) transfection. Results and conclusion: Of the 11 ginsenosides tested, 20S-protopanaxadiol (PPD) showed the most potent cytotoxic activity in both LX-2 cells and primary activated HSCs. Oxidative stress-mediated apoptosis induced by 20S-PPD was blocked by N-acetyl-$\text\tiny L$-cysteine pretreatment. In addition, 20S-PPD concentration-dependently increased the phosphorylation of AMPK, and compound C prevented 20S-PPD-induced cytotoxicity and mitochondrial dysfunction. Moreover, 20S-PPD increased the phosphorylation of liver kinase B1 (LKB1), an upstream kinase of AMPK. Likewise, transfection of LX-2 cells with LKB1 siRNA reduced the cytotoxic effect of 20S-PPD. Thus, 20S-PPD appears to induce HSC apoptosis by activating LKB1-AMPK and to be a therapeutic candidate for the prevention or treatment of liver fibrosis.

• Journal of Ginseng Research
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• v.45 no.4
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• pp.465-472
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• 2021

Background: Ginseng can help regulate brain excitability, promote learning and memory, and resist cerebral ischemia in the central nervous system. Ginsenosides are the major effective compounds of Ginseng, but their protein targets in the brain have not been determined. Methods: We screened proteins that interact with the main components of ginseng (ginsenosides) by affinity chromatography and identified the 14-3-3 ζ protein as a potential target of ginsenosides in brain tissues. Results: Biolayer interferometry (BLI) analysis showed that 20(S)-protopanaxadiol (PPD), a ginseng saponin metabolite, exhibited the highest direct interaction to the 14-3-3 ζ protein. Subsequently, BLI kinetics analysis and isothermal titration calorimetry (ITC) assay showed that PPD specifically bound to the 14-3-3 ζ protein. The cocrystal structure of the 14-3-3 ζ protein-PPD complex showed that the main interactions occurred between the residues R56, R127, and Y128 of the 14-3-3 ζ protein and a portion of PPD. Moreover, mutating any of the above residues resulted in a significant decrease of affinity between PPD and the 14-3-3 ζ protein. Conclusion: Our results indicate the 14-3-3 ζ protein is the target of PPD, a ginsenoside metabolite. Crystallographic and mutagenesis studies suggest a direct interaction between PPD and the 14-3-3 ζ protein. This finding can help in the development of small-molecular compounds that bind to the 14-3-3 ζ protein on the basis of the structure of dammarane-type triterpenoid.

• Journal of Ginseng Research
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• v.46 no.5
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• pp.666-674
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• 2022

Background: Ginsenosides and their metabolites have antidepressant-like effects, but the underlying mechanisms remain unclear. We previously identified 14-3-3 ζ as one of the target proteins of 20 (S)-protopanaxadiol (PPD), a fully deglycosylated ginsenoside metabolite. Methods: Corticosterone (CORT) was administered repeatedly to induce the depression model, and PPD was given concurrently. The tail suspension test (TST) and the forced swimming test (FST) were used for behavioral evaluation. All mice were sacrificed. Golgi-cox staining, GSK 3β activity assay, and Western blot analysis were performed. In vitro, the kinetic binding analysis with the Biolayer Interferometry (BLI) was used to determine the molecular interactions. Results: TST and FST both revealed that PPD reversed CORT-induced behavioral deficits. PPD also ameliorated the CORT-induced expression alterations of hippocampal Ser9 phosphorylated glycogen synthase kinase 3β (p-Ser9 GSK 3β), Ser133 phosphorylated cAMP response element-binding protein (p-Ser133 CREB), and brain-derived neurotrophic factor (BDNF). Moreover, PPD attenuated the CORT-induced increase in GSK 3β activity and decrease in dendritic spine density in the hippocampus. In vitro, 14-3-3 ζ protein specifically bound to p-Ser9 GSK 3β polypeptide. PPD promoted the binding and subsequently decreased GSK 3β activity. Conclusion: These findings demonstrated the antidepressant-like effects of PPD on the CORT-induced mouse depression model and indicated a possible target-based mechanism. The combination of PPD with the 14-3-3 ζ protein may promote the binding of 14-3-3 ζ to p-GSK 3β (Ser9) and enhance the inhibition of Ser9 phosphorylation on GSK 3β kinase activity, thereby activating the plasticity-related CREBeBDNF signaling pathway.

• Journal of Ginseng Research
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• v.47 no.5
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• pp.662-671
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• 2023

Background: 20(S)-protopanaxadiol (PPD), a ginsenoside metabolite, has prominent benefits for the central nervous system, especially in improving learning and memory. However, its transcriptional targets in brain tissue remain unknown. Methods: In this study, we first used mass spectrometry-based drug affinity responsive target stability (DARTS) to identify the potential proteins of ginsenosides and intersected them with the transcription factor library. Second, the transcription factor PURA was confirmed as a target of PPD by biolayer interferometry (BLI) and molecular docking. Next, the effect of PPD on the transcriptional levels of target genes of PURA in brain tissues was determined by qRT-PCR. Finally, bioinformatics analysis was used to analyze the potential biological features of these target proteins. Results: The results showed three overlapping transcription factors between the proteomics of DARTS and transcription factor library. BLI analysis further showed that PPD had a higher direct interaction with PURA than parent ginsenosides. Subsequently, BLI kinetic analysis, molecular docking, and mutations in key amino acids of PURA indicated that PPD specifically bound to PURA. The results of qRT-PCR showed that PPD could increase the transcription levels of PURA target genes in brain. Finally, bioinformatics analysis showed that these target proteins were involved in learning and memory function. Conclusion: The above-mentioned findings indicate that PURA is a transcription target of PPD in brain, and PPD upregulate the transcription levels of target genes related to cognitive dysfunction by binding PURA, which could provide a chemical and biological basis for the study of treating cognitive impairment by targeting PURA.

• Tuberculosis and Respiratory Diseases
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• v.61 no.3
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• pp.239-247
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• 2006

Background: Culture filtrate proteins secreted by mycobacteria are thought to play an important role in inducing protective immunity and to develop new methods for diagnosing tuberculosis. Methods: A culture filtrate protein of M. avium that was strongly reactive with goat antiserum against M. intracellulare was constructed. Its homologous protein (TB-14) in M. tuberculosis was cloned, expressed and purified. The inductions of IFN-${\gamma}$ stimulated with $10{\mu}g$ of TB-14 recombinant protein and $10{\mu}g$ PPD were estimated by using whole bloods from seven PPD (-) subjects, seven PPD (+) healthy volunteers and nine tuberculosis patients. Results: M. avium culture filtrate protein was confirmed as a hypothetical protein that was termed contig 116. A novel 14-kDa recombinant protein (TB-14) of M. tuberculosis was composed of 148 amino acids, including 30 amino acids of the signal peptide, and it showed 78% homology with M. avium. In the PPD (+) healthy volunteers, recombinant TB-14 protein strongly induced the secretion of IFN-${\gamma}$ in whole blood cultures. Conclusion: These results suggest that TB-14 recombinant protein might play an important role in inducing cell-mediated immunity against tuberculosis. Furthermore, TB-14 protein antigen and its antiserum will be available for the development of new diagnostic tools for tuberculosis.

• Bulletin of the Korean Chemical Society
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• v.27 no.8
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• pp.1219-1221
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• 2006